The present invention relates to a cloned "basic helix loop helix -PER-ARNT-AhR-SIM" (bHLH-PAS) protein that is a juvenile hormone receptor (JHR), bHLH-PAS/JHR. In particular, this invention is directed to a bHLH-PAS/JHR gene isolated from Drosophila, termed the methoprene-tolerant (met) gene (Met-JHR). The present invention also is directed to in vitro and in vivo methods for screening insecticides using recombinant bHLH-PAS/JHRs. The present invention is further directed to methods for isolating polynucleotides encoding bHLH-PAS/JHRs from various insect species.
Worldwide insect damage to food and fiber costs billions of dollars annually. Although chemical insecticides are still the primary means of insect control, the use of chemicals has several drawbacks including high cost of discovery, potential environmental damage, and negative public opinion. One promising group of insecticides consists of analogues of insect hormones, such as juvenile hormone. Since vertebrates do not make juvenile hormone (JH), insecticides targeted to the JH system are highly toxic to certain insects, and have shown an extraordinary degree of environmental safety.
Juvenile hormones comprise a family of hormones that are secreted by the corpus allatum, and that play a role in a variety of critical functions in insects, including development, reproduction, and morphological differentiation. Riddiford, "Hormone Action at the Cellular Level," in COMPREHENSIVE INSECT PHYSIOLOGY, BIOCHEMISTRY AND PHARMACOLOGY, VOLUME 8, Kerkut et al. (eds.), pages 37-84 (Pergamon Press 1985); Nijhout et al., Q. Rev. Biol. 57:109 (1982). These hormones affect development in some insects by maintaining the larval stage and inhibiting metamorphosis. In adult insects, JH is involved in the regulation of reproductive physiology. Koeppe et al., "The Role of Juvenile Hormone in Reproduction," in COMPREHENSIVE INSECT PHYSIOLOGY, BIOCHEMISTRY AND PHARMACOLOGY, VOLUME 8, Kerkut et al. (eds.), pages 165-203 (Pergamon Press 1985).
The action of JH is mediated by at least several types of JH binding proteins: a hemolymph carrier protein, a cell membrane bound receptor, and an intracellular receptor. The transport of JH to target tissues is believed to be accomplished by proteins in the hemolymph which bind with the hormone. Hammock et al., Pestic. Biochem. Physiol. 7:517 (1977); Goodman et al., "Juvenile Hormone Cellular and Hemolymph Binding Proteins," in COMPREHENSIVE INSECT PHYSIOLOGY, BIOCHEMISTRY AND PHARMACOLOGY, VOLUME 7, Kerkut et al. (eds.), pages 491-510 (Pergamon Press 1985). These JH binding proteins are thought to play roles both in the transport of JH and in the protection of JH from hemolymph esterases. Goodman et al., Am. Zool. 14:1289 1974; Kramer et al., J. Biol. Chem. 251:4979 (1974). Membrane bound receptors are known to bind ligand extracellularly and transmit a signal intracellularly. Wyatt et al. Adv. Insect Physiol. 26:1 (1996).
Cytosolic proteins that bind JH have been identified in numerous JH target tissues from a variety of insects. Van Mellaert et al., Insect Biochem. 15:655 (1985); Klages et al., Nature 286:282 (1980); Engelmann et al., Insect Biochem. 17:1045 (1987); Wisniewski et al., FEBS Lett. 171:127 (1984). One of the inventors, Thomas G. Wilson, directed a research team that identified a cytosolic juvenile hormone-binding protein in Drosophila melanogaster that is characterized by saturable, high-affinity binding specific for JH III. Shemshedini et al., J. Biol. Chem. 265:1913 (1990). Shemshedini et al. also demonstrated for the first time in any insect a correlation between the binding of JH to the cytosolic protein and a biological response to the hormone. Interference with the binding of JH to cognate intracellular receptors, therefore, would inhibit physiological functions dependent upon the hormone.
Until recently, novel insecticides that interfere with JH action were primarily discovered by an almost random testing of thousands of chemical compounds for efficacy against insects. This bioassay approach is slow and expensive since a group of test insects would have to be treated with various doses of each test compound, and, typically, finding compounds that are effective is exceedingly rare.
Accordingly, a need exists for an efficient method for testing insecticides targeted for the JH system.